The present invention is a sequence modification of the H7 hemagglutinin glycoprotein of the Influenza A/Shanghai/2/2013 H7 sequence.
The discordant immunogenicity of vaccines developed for two distinct emerging influenza A viruses (IAV), 2009 pandemic H1N1 (A(H1N1)pdm09) and H7N9 avian influenza (H7N9), provided an opportunity to evaluate the role of T cells in the development of effective humoral immune response. For example, although A(H1N1)pdm09 was highly transmissible and spread to more than 200 countries within 12 months of emergence due to the lack of pre-existing antibodies, morbidity and mortality due to the A(H1N1)pdm09 influenza were lower than expected, presumably due to pre-existing T cell responses among individuals exposed to or vaccinated with seasonal A(H1N1) strains. H7N9's emergence in China in 2013 was associated with much higher lethality. Due to concerns about its lethality and pandemic potential, H7N9 vaccines were prioritized for production, and vaccines were developed.
Influenza vaccines can call upon memory T cells to generate protective immunity and stimulate antibody response in the absence of adjuvants; thus, usually only one vaccination is required to generate protective immunity to seasonal influenza strains. Conventional recombinant H7 hemagglutinin vaccines produced to address the re-emergence of avian-origin H7N9 influenza (for which cross-reactive humoral immunity is presumed to be absent) in China have proven to have poor efficacy compared to other subunit and seasonal influenza vaccines. In stark contrast with A(H1N1)pdm09, un-adjuvanted H7N9 vaccines were poorly antigenic and vaccination with un-adjuvanted H7N9 hemagglutinin (HA) resulted in hemagglutination inhibition (HI) seroconversion rates of only 6% and 15.6% in Phase I clinical trials (as compared to 89% for similar un-adjuvanted A(H1N1)pdm09 subunit vaccines). Clinical trials of these vaccines have required the use of adjuvant to increase the antigenicity of these vaccines to acceptable standards, however, adjuvants are not used in standard seasonal influenza vaccines in the United States. Even when two doses of H7N9 vaccine were administered with adjuvant to generate new memory T helper cells to the novel virus, only 59% of subjects sero-converted in a recent Phase II clinical trial. The development of neutralizing antibodies to H7N9 is also delayed in H7N9-infected humans when compared to the typical immune response to other IAV infections and IgG avidity to H7N9 HA is significantly lower. In clinical trials of other H7 subtypes, an attenuated H7N1 vaccine elicited low HI titers, and an inactivated subunit H7N7 vaccine was poorly immunogenic.
H7 HA appears to be uniquely non-antigenic. The observed human antibody response to a related H7 HA in the H7N7 outbreak in 2003 in the Netherlands was also diminished in HI titer. Taken together, these studies suggest that adaptive immune responses to H7N9 infection may be diminished and delayed, even in the context of natural infection.
CD4+ T cells provide help to B cells, supporting isotype conversion and affinity maturation; thus, diminished and delayed antibody responses to H7 HA suggest that T cell help was limited or abrogated. There are fewer CD4+ T helper epitopes in the H7N9 sequences than in other IAV. Similar patterns of epitope deletion have been observed in chronic (‘hit-and-stay’) viruses that have adapted to the human host, such as Epstein Barr virus (EBV) and Herpes simplex virus (HSV), but not in acute (‘hit-and-run’) viruses Immune escape mediated by epitope deletion is a well-established mechanism of viral pathogenesis for human immunodeficiency virus (HIV) and hepatitis C virus (HCV), but this escape mechanism has not been previously described for influenza.
Another means by which H7N9 may minimize host response is to adopt ‘immune camouflage’, a new mechanism of immune escape identified by our group. T cell epitopes derived from pathogens that have high T cell receptor (TCR) ‘cross-conservation’ with human sequences can be identified using JanusMatrix (EpiVax, Providence, R.I., USA), an algorithm that compares TCR-facing patterns of CD4+ T cell epitopes to sequence patterns present in the human genome. JanusMatrix is a homology analysis tool that considers aspects of antigen recognition that are not captured by raw sequence alignment. Commensal viruses contain a significantly higher number of these JanusMatrix-defined ‘human-like’ T cell epitopes than viruses that do not establish chronic infections in humans.
HCV contains an epitope that is highly cross-conserved with self and significantly expands T regulatory cells (Tregs) in vitro. T cells that respond to this peptide exhibit markers that are characteristic of Tregs and actively suppress bystander effector T cell responses in vitro. The striking difference between chronic-disease viruses, which appear to have many such epitopes, and acute-disease, pathogenic viruses, suggests that immune camouflage may be an important method by which certain human pathogens escape adaptive immune response.
Pre-existing heterotypic T cell memory specific for epitopes contained in the new flu strain obviate the need for adjuvants and effective antibody titers may develop following a single dose as was observed for A(H1N1)pdm09 (Greenberg M E et al., N. Engl. J. Med., 361:2405-13, 2009). While T cell epitopes that recall pre-existing immunity may help protect against multiple viral subtypes as was observed for A(H1N1)pdm09 influenza (Laurie K L et al., J. Infect. Dis., 202:1011-20, 2010), epitopes that resemble host sequences may be detrimental to immunity.
In a retrospective analysis of published viral epitopes in a large epitope database, greater human cross-conservation was associated with absent or regulatory T cell responses (He L et al., BMC Bioinformatics, 15:S1, 2014). Taken together, these findings demonstrate that certain human pathogens may evolve to contain T cell epitopes in their proteomes that resemble important human regulatory T cell epitopes (‘immune camouflage’).
The T cell epitope profile of H7N9 (few effector T cell epitopes and many cross-conserved epitopes) is much closer to these ‘hit-and-stay’ viruses than viruses that ‘hit-and-run’. Although human-to-human transmission of H7N9 is rare, the virus has been noted to have a ‘mammalian signature’. Cases of limited human-to-human transmission have been reported (Gao H N et al., Int. J. Infect. Dis., 29C:254-8, 2014). Human-to-human transmission of H7N9 may occur more frequently than suspected making it harder to detect due to low titers of antibody. The discovery of human-like epitopes in the H7N9 proteome raises an important question about the origin and evolution of H7N9 and the duration of its circulation in human beings or other mammals.
The H7N9 genome (made publicly available on the GISAID website on Apr. 2, 2013) was analyzed using an immunoinformatics toolkit. The analysis indicated that the H7 HA had fewer than expected T-cell epitopes and would be poorly immunogenic.
Accordingly, a need remains for influenza vaccines with greater efficacy to address the re-emergence of avian-origin H7N9 influenza in China without the use of adjuvant to increase the antigenicity.